Transcriptional silencer protein NRF

ABSTRACT

NRF is a novel inhibitory transcription factor binding to specific DNA sequences and silencing transcriptional activity of proximal DNA-binding activators, e.g. NF- K B binding sites.  
     NRF is a modulator protein of NF- K B family members controlling genes of significant biomedical importance such as those encoding inflammatory cytokines, MHC proteins, cell adhesion molecules, and viruses. Based on this it represents a molecular target in the development of novel anti-inflammatory therapies for a variety of pathologic disorders such as ischemia, hemorrhagic and septic shock, allograft rejection, bacterial meningitis, acute airway inflammation and the pulmonary complications induced by cardiopulmonary bypass. Furthermore, this might apply for certain cancers and other diseases.  
     NRF as a target for drugs (obtained e.g. by IITS) is protected. Agonists as well as antagonists of NRF are also protected. Further applications such as the use of NRF protein and its fusion proteins or NRF DNA sequence in sense or antisense orientation in gene therapy are also protected.

[0001] This is a continuation of International Application No.PCT/EP98/04661 filed Jul. 24, 1998 (International Publication No. WO99/05269 dated Feb. 4, 1999). The respective disclosures ofInternational Application No. PCT/EP98/04661 and InternationalPublication No. WO 99/05269 are incorporated herein by reference.

[0002] The invention concerns the transcriptional silencer protein NRFwhich is a novel inhibitory transcription factor, and several relatedsubject matters. The background of the invention is as follows.

[0003] NF-_(K)B/rel Protein

[0004] The family of NF-_(K)B/rel transcription factors regulates avariety of promoters through specific DNA-binding sites.NF-_(K)B/rel-binding sites act as weak constitutive enhancers. However,many promoters which contain NF-_(K)B/rel-binding sites do not show baselevel activity. This is explained by the existence of silencer elements.Further to the constitutive enhancing activity of the NF-_(K)B bindingsites, many inducers like viruses, TNF-A or PMA induce a signallingcascade that increases the activity of the NE-_(K)B enhancerstransiently. These inducers lead to a transient inactivation of IkB, thecytoplasmic inhibitor of certain NF-_(K)B members. This results in anuclear translocation of the prototype NF-_(K)B (a heterodimer of p50and p65) and the activation of the above mentioned target genes bybinding and activation of transcription.

[0005] The nuclear factor NF-_(K)B rel family is involved in theregulation of a number of genes which contribute to physiologicalactivities, like inflammation and cell growth. Inflammatory pathologyand cancer are often associated with the disregulation of theses genes,raising the possibility that the initiation of multiple pathologicprocesses is due to NF-_(K)B/rel-mediated transactivation. Inhibitors ofNF-_(K)B activation may therefore have broad applications as noveltherapeutics in human diseases. Natural repression mechanisms might playan important part in the control of disregulated NF-_(K)B/rel activity.

[0006] The IFN-β Negative Regulatory Element (NRE)

[0007] An example of a repression mechanism was found in the control ofmammalian Interferon-β (IFN-β) gene expression. IFN-β genes areabsolutely silent but can be transcriptionally activated in nearly alldifferentiated type of cells by viruses or double-stranded RNA.

[0008] In this promoter several positive regulatory domains (PRDs) areassembled within less than loo base pairs. The PRDII, which represents abinding site for NF-_(K)B/rel proteins and PRDI to which members of theIRF-family can bind are responsible for a basal expression level (1, 9,14, 18, 38). For full activity of the IFN-β promoter further PRDsequences are required.

[0009] A minimal virus response element (VRE) was identified. Itcontains PRDI, PRDII and an extended negative regulatory domain (NRD).The NRD was found to repress a basal transcriptional activity in theabsence of inducers (10, 11). Within this NRD, an 11 base pair elementacts as a negative regulatory element (NRE) of the PRDII sequence.Although this NRE is physically overlapping with PRDII, it can act as aposition-independent silencer of PRDII. (24)

[0010] NREs in Other Promoters

[0011] Examination of NF-_(K)B/rel-binding sites containing promotersfor the presence of NRE-related sequences and functions revealed severalelements with a loose sequence relationship to the IFN-β NRE.

[0012] In the HIV-1 promoter, two sequences with homology to the IFN-βNRE were found in a region which was functionally defined as a region ofnegative regulation for HIV-1 transcription (3, 22). Saksela andBaltimore (1993) have described a negatively acting element (termed_(K)NE) immediately upstream of the NF-_(K)B-binding site in the Ig_(K)intronic enhancer. The core of this 27-bp _(K)NE sequence shows a highhomology to the IFN-β NRE. A negative regulatory element was shown toexist in the IL-2 receptor α chain promoter (33) also exhibiting somesequence similarity to the IFN-β NRE. Homology to NRE in the HTLV-Ipromoter was also found. The region in which this sequence is locatedcooperates positively with the 21-bp enhancer upon Tax proteinactivation (34). The inducibility of these promoters involves theactivation of NF-_(K)B/rel binding sequences.

[0013] The NRE-related sequences contained within the promoters of HIV-1and HTL V-1 and the IL-2R-α gene constitute functionally relatedsilencer elements which repress the constitutive enhancing activity ofNF-_(K)B/rel-binding sites from these promoters. Thus, NREs represent anew class of transcriptional repressor sequences with a silencingactivity on the constitutive activity of NF-_(K)B/rel binding sites.

[0014] All NREs show similar properties with respect to binding ofproteins from nuclear extracts, however, with distinguishableaffinities. The distinct affinities reflect the silencing capacity ofthe NREs. The NRE-mediated silencing effect is relieved byenhancer-specific inducers like viruses, TAX or PMA. Despite thishomology, the NF kB/rel DNA-binding sites from HIV-1-LTR and IFN-βpromoter exhibit significant differences. These differences are based onthe sequence specificities of the NF-_(K)B/rel-binding sites, but not onthe sequences of the NREs.

[0015] Common Features of NRES:

[0016] The common features exerted by the presently known five NREs are:sequence homology, short length (11-13 bp), distance andposition-independent action, specific interaction withNF-_(K)B/rel-binding sequences and indistinguishable binding patterns ofnuclear factors. A considerable number of other NREs in various genes isexpected to exist.

[0017] For example, sequence comparisons show NRE homology sequenceswithin the promoters of the cell adhesion molecules ELAM-1 and CAM-1. Anegatively acting element (termed kNE) with homology to the NRE coresequence was described immediately upstream of the NF-_(K)B-binding sitein the Igk intronic enhancer (27).

[0018] Target Sequence Specificity of NRE-function:

[0019] The NREs do not act on the basal transcription machinery. (24) Upto now, only the NRE silencing of NF-_(K)B/rel enhancers has been found.However, other activating sequences may also be silenced by the NREs.This is supported by the identification of a silencer from the gastrinpromoter (39) which is highly homologous to the described NREs. Thegastrin promoter does not contain NF-_(K)B/rel or related binding sites,assuming that other enhancer(s) interact with the gastrin silencer (37).

[0020] Proteins Binding to NREs:

[0021] Sequence and functional homology of the NREs from differentsources suggest that the binding factors to these NREs are distinct oridentical.

[0022] In EMSA the IFN-β NRE (N) sequence is retarded to give two majorcomplexes (bands), the faster one having a higher binding affinity thanthe slower migrating complex (24). All functional NRE sequences areretarded in exactly the same manner, giving rise to the two complexes.The ability to form indistinguishable complexes suggests that thefactors binding to these oligonucleotides are identical. This wasfurther confirmed by cross-competition experiments. All NREs competewith each other in the same way as by themselves. However, competitiondata also demonstrate that affinity within the different complexesdiffers. The highest affinity is exhibited by IFN-β NRE, followed by theIL-2 Rec α NRE. NREs from both HIV-1 and HTLV-I show a clearly loweraffinity. The affinities of NREs to nuclear proteins roughly reflecttheir silencing capacity to the NF-_(K)B/rel enhancer.

[0023] UV-crosslinking data suggested that the proteins would havemolecular weight(s) of about 100 KDa (24). The currently publishedexperiments do not allow a determination of the number of factors thatare involved in the NRE specific silencing function(s).

[0024] Known NF-_(K)B-repressors:

[0025] Recently, a nuclear NF-_(K)B/rel inhibitor was described. Thisfactor inhibits DNA-binding of p50/p65 heterodimers in Adenovirustransformed cells (21). Obviously, this repressor acts by suppressingthe induced NF-_(K)B-enhancer activity and is therefore distinct to thefactor(s) which repress the constitutive NF-_(K)B-enhancer activity.

[0026] A Drosophila 43 KDa HMG1 protein called DSP1 (dorsal switchprotein) was described. This protein inhibits the Dorsal enhancer bybinding to a proximally located sequence (17). Furthermore, DSP-1represses NF-_(K)B/rel-site mediated enhancement in mammalian cells. Thehuman homologue to DSP-1 which was believed to act as the IFN-β specificsilencing protein, has not yet been described.

[0027] The viral transactivator Tax of HTLV-1 is able to restore theactivity of the HIV-1 NF-_(K)B enhancer silenced by any NRE. Similar tothis observation, Salvetti et al. (29) described the repression of anNF-_(K)B/rel binding site in the human vimentin promoter by a negativeelement which would be relieved by Tax expression. Tax transactivatesseveral promoters through the induction of NF-_(K)B/rel proteins bynuclear translocation of cytoplasmatic dimers and de novo synthesis ofc-rel (16). It has been shown that Tax is able to induce nucleartranslocation of NF-_(K)B/rel proteins retained in the cytoplasm throughinteraction with p 105 or p 100 (19, 23). Induction of NF-_(K)B/relenhancing activity by the Tax protein would result in masking theinhibitory effect exerted by the NREs. The unresponsiveness of the IFN-βand HTLV-1 NF-_(K)B enhancers to Tax expression and thus the unalteredrepression by NREs may be due to the sequence differences of theinvestigated enhancers. Such differences in binding and transactivationof the known NF-_(K)B/rel dimers are well documented (20).

[0028] Release from Repression:

[0029] The repressive effect of the IFN-β NRE on PRDII cannot beeliminated by viral infection although this leads to an induction ofNF-_(K)B binding activity (24). The inducible derepression of PRDII isdependent on the interaction with PRDI, a binding site for IRF-proteins.Similarly, induced NF-_(K)B/rel activity due to viral infection is notsufficient to activate the HTLV-1 enhancer. Depending on the NF-_(K)Benhancer element and the inducing agent, derepression requires anadditional activator. The concerted action of coactivators or theinduction of a particular set of NF-_(K)B/rel binding proteins aresufficient for releasing the repression.

[0030] Virus induction does not affect the negative activity of the NREon isolated PRDII. However, a 28 base pair fragment containing PRDI,PRDII and NRE functions as a minimal VRE indicating that it is thecooperative effect of PRDI and PRDII which is responsible for overcomingthe NRE function after virus infection. These properties together withelectromobility shifts and DNA-crosslinking data indicate that theproteins being responsible for the silencing effect are still bound tothe NRE after transcriptional induction by virus infection. It wasspeculated that the silencing effect of NRE-binding factor(s) might bedue to a masking of the NF-_(K)B/rel activator or to ‘locking’ of thebasal transcriptional complex for NF-_(K)B/rel activation. It wasfurther speculated that replacement, post-transcriptional or stericalterations of the factors bound to the PRDs could eliminate thenegative activity of the silencer protein(s) (24).

[0031] Apart from the activation mechanism, NF-_(K)B sequences exhibit abasal constitutive activity. Most probably, this background activity ismaintained by the binding of NF-_(K)B/rel proteins which are notcytoplasmatically retained by I-_(K)B, e. g. p50 dimers. This basalactivity is repressed in a number of NF-_(K)B promoters including thoseregulating IFN-β, IL-2 receptors-α chain.

[0032] According to one embodiment the invention concerns a ssDNA

[0033] (a) having the sequence according to FIG. 1B or

[0034] (b) having the sequence according to FIG. 1B wherein

[0035] (i) at positions 984 to 1077 and

[0036] (ii) at positions 1897 to 1979

[0037] the nucleotides shown in FIG. 1B are replaced by those shownbelow them in the second row or

[0038] (c1) having the same number of nucleotides as the ssDNA accordingto (a) or

[0039] (c2) having a reduced number of nucleotides compared with thessDNA according to (a)

[0040] wherein the ssDNA according to (c1) and (c2) is hybridizable withthat according to (a) and/or (b).

[0041] A ssDNA according to the invention may comprise or have thenucleotide region

[0042] (i) of from position 654 to position 1817 or

[0043] (ii) of from position 1518 to 1817 (DNA binding domain=DBD) or

[0044] (iii) of from position 654 to position 1526 (silencer domain) or

[0045] (IV) of from position 1 to position 653 according to FIG. 1B orssDNA

[0046] (v-i) having the same or a reduced number of nucleotides comparedwith the ssDNA according to (i) or

[0047] (v-ii) having the same or a reduced number of nucleotidescompared with the ssDNA according to (ii) or

[0048] (v-iii) having the same or a reduced number of nucleotidescompared with the ssDNA according to (iii) or

[0049] (v-iv) having the same or a reduced number of nucleotidescompared with the ssDNA according to (iv)

[0050] wherein the ssDNA according (v-i), (v-ii), (v-iii) and (v-iv) ishybridizable with that according to (i), (ii), (iii) and (iv)

[0051] As regards a ssDNA according to (iv) or (v-iv) or a dsDNA or aRNA corresponding thereto, the following background is given. NRF mRNAhas an extraordinarily long 5′untranslated region of 654 nucleotidescontaining several open reading frames. In principle, mRNAs whichcontain unusually long leader sequences with multiple upstream readingframes are good candidates for initiating translation viacap-independent internal ribosome binding mechanism (Sachs et al.,1997). The cap-independent internal initiation model was initiallyproposed in picornaviral mRNAs. These internal ribosome entry sites(IRES) have been successfully removed from their viral setting andlinked to unrelated genes to produce polycistronic RNAs. A few cellularmRNAs have also been found to contain IRESs. As described so far,cellular IRESs display low efficiency in directing translation byinternaL initiation. The strength of translation initiation from IRESsis equal or weaker when compared to cap-dependent translationinitiation.

[0052] Another embodiment of the invention concerns a ssDNA which ischaracterized in that it is complementary to a ssDNA as describedbefore.

[0053] Hybridization condition for hybridizable ssDNAs (c1), (c2) or(iv) as defined before are, for example, at a temperature of at least25° C. and a 1 M sodium chloride concentration.

[0054] According to another embodiment the invention concerns the dsDNAconsisting of a ssDNA as described above and its complementary strand.

[0055] According to another embodiment the invention concerns a RNA.

[0056] (a) having a sequence corresponding to that of a DNA according tothe invention as described above or

[0057] (b) having a sequence corresponding to that of a RNA according to(a) but in anti-sense or

[0058] (c) being a degradation product o a RNA according to (a) or (b)being degraded in a manner known per se.

[0059] According to another embodiment the invention concerns a vectorcomprising a dsDNA as described. The vector may comprise

[0060] (i) a dsDNA consisting of a ssDNA as described in paragraphs (i),(ii) and (iii) before and a complementary strand or

[0061] (ii) a ssDNA according to paragraphs (i), (ii) and (iii) asdescribed before and a complementary strand coding the same amino acidas a dsDNA according to (i) but comprised by the vector in antisensedirection.

[0062] A vector according to the invention may be used for thetransformation of cells and organisms for transient or for permanentexpression of a protein encoded by the dsDNA comprised by the vector.

[0063] Another embodiment of the invention concerns a protein encoded bya ssDNA or a dsDNA as described before, optionally fused with anotherfunctional protein or one or more functional fragments thereof. Asregards these functional fragments, they may be fused with the proteinaccording to the invention as interspiced fragments. Of course, theprotein according to the invention may be an unfused protein.

[0064] Another embodiment of the invention concerns a protein (dominantnegative mutant) which can be obtained by

[0065] (a) mutating the nucleotide sequence of a ssDNA according to theinvention in a manner known per se,

[0066] (b) expressing the mutated ssDNA (ssDNAs) in a manner known perse

[0067] (c) subjecting the expression product(s) to a competing test forinhibition of transcription with a protein encoded by the unmodifiedssDNA (starting ssDNA) and

[0068] (d) isolating a protein which acts as a dominant negative mutantof the protein encoded by the unmodified ssDNA.

[0069] A protein which represses the human IFN-β promoter was postulatedearlier. However, the properties of NRF and the effects exerted uponoverexpression of its sense and antisense RNA are unexpected because

[0070] crosslinking analysis revealed not one but at least two proteinsof about 100 kDa molecular mass and

[0071] NRF has no homology to teh DSP-1 protein which was thought to bethe Drosophila homologue to the IFN-β repressor (17).

[0072] The protein(s) according to the invention encoded by the humangene has (have) the following properties:

[0073] It binds specifically DNA-sequences which are identical orrelated to the NRE-motif. The NREs are contained in a number ofpromoters of human genes.

[0074] It affects transcription of a number of cellular and viral genes,e. g. it represses the background activity of NF-_(K)B/rel-bindingenhancer elements. By these properties, it constitutively represses theactivity of a number of cellular genes.

[0075] Inactivation of endogenous NRE expression leads to the inductionof cellular genes, i. g. the IFN-β gene.

[0076] NRF can be regarded as a modulator protein of NF-_(K)B familymembers controlling genes of significant biomedical importance such asthose encoding inflammatory cytokines, MHC proteins, cell adhesionmolecules, and viruses. Based on this it represents a molecular targetin the development of novel anti-inflammatory therapies for a variety ofpathologic disorders such as ischemia, hemorrhagic and septic shock,allograft rejection, bacterial meningitis, acute airway inflammation andthe pulmonary complications induced by cardiopulmonary bypass.Furthermore, this might apply for certain cancers and other diseases.

[0077] Another embodiment of the invention concerns a use of

[0078] (i) a ssDNA according to the invention or

[0079] (ii) a dsDNA according to the invention or

[0080] (iii) a vector according to the invention or

[0081] (iv) a protein according to the invention

[0082] (a) for identifying and developing agonists and antagonists ofNRF-functions,

[0083] (b) for the development of improved antisense NRF and ribozymes,

[0084] (c) for the detection and diagnosis of transient or permanentregulatory disorders of NF_(K)B/rel- and/or NRF-regulated physiologicalpatterns in animals and humans or

[0085] (d) for therapy development and treatment of diseases, especiallyrheumatoid, arthritis, inflammations, infectious diseases, tumors and/orgenetic diseases, or

[0086] (e) for gene therapy in animals and humans.

[0087] Finally, another embodiment of the invention concerns a use of aRNA according to the invention having a sequence according to that of aDNA according to (iv) oder (v-iv) as indicated above.

[0088] Said use can be as IHRES element in a polycistronic expressionvector for application in an eucaryotic cell or a transgenic animal.

[0089] Finally, said use can be as translational enhancer in amonocistronic or a polycistronic expression vector for application in aneucaryotic cell or a transgenic animal.

[0090] The following FIGS. 1 to 9 and the following examples explain theinvention in greater detail.

EXAMPLE 1

[0091] 1. Cloning of the Human and Mouse NRE Binding Factor (NegativeRegulatory Factor):

[0092] A) The method relies on the expression of human cDNA inserts fromHeLa cells in bacteriophage lambda gtll. Fusion protein adsorbed ontonitrocellulose filters (NC) is probed with radioactive, double-strandedNRE-sequence as a ligand; NRE cf. Nourbakhsh et al. in EMBO J., 12(1993) 451-459. Specific NRE-binding signals were detected on filtersand corresponding bacteriophage plaques were isolated. SpecificDNA-binding signals were detected on duplicate filters probed eitherwith NRE-sequence or mutant NRE-sequence.

[0093] A cDNA clone coding for a 44 kd protein was detected with highspecificity for NRE-sequence. Bacterial cells were infected withdistinct number of the recombinant bacteriophage carrying cDNA of 44 kdprotein and duplicate filters were probed with labeled DNA as indicated.The filters were exposed to X-ray film overnight, generatingautoradiographic images (FIG. 1A).

[0094] B) The human NRF cDNA was used as hybridization probe to screenfor homologous sequences in a cDNA-bank from mouse embryos (day 11) atreduced stringency. Identified clones were isolated, characterized andsequenced. FIG. 1B shows the sequence alignment of human (middle line)and murine (underneath) NRF cDNA. The protein coding region of human NRFcDNA is given by indicated amino acid sequence above the human cDNAsequence.

[0095] 2. Structure of the Human Negative Regulatory Factor (NRF)

[0096] Two mRNAs with different 3′untranslated regions were identified,coding for NRF in human cells. The size of the NRF mRNAs is 2.8 and 3.8kb (FIGS. 3 and 4). The coding region of mRNA is indicated by an openbar in FIG. 2. In the same figure, the protein sequence of NRFconsisting of 388 aa is demonstrated by a dark bar. The silencer domainof NRF consisting of first 291 aa contains two different zinc-fingersindicated by bubbles. The DNA binding domain of NRF (DBD) is within 100amino acids of the C-terminal end and contains a helix-loop-helix motif(FIG. 2).

[0097] 3. NRF is Constitutively Expressed:

[0098] The expression level of NRF was determined by Northern blotanalysis using poly(A)-RNA from HeLa cells. In FIG. 3, two differentmRNA corresponding to NRF are indicated (top). Both mRNAs areconstitutively expressed and their level of expression is not alteredafter treatment of the cells with Newcastle disease virus. Interferon-βmRNA (middle) shows a typical induction of the cells. Actin mRNAindicates equal amounts of RNA on each lane (obtained byrehybridization).

[0099] 4. NRF is Ubiquitously Expressed:

[0100] The expression level of NRF in different human tissues wasdetermined by Northern blot analysis. Two different mRNA correspondingto NRF are indicated on the top. Actin mRNA is indicated showing anequal amount of RNA on each lane (FIG. 4).

[0101] 5. NRF Binds to NRE Regulated Promoters in vivo:

[0102] DNA-binding activity of NRF in vivo was tested using constructsencoding either NRF or a chimaeric protein consisting of full length NRFand the VP16 activating domain from Herpes simplex virus. Expression ofthese chimaeric proteins were carried out in murine cells habouringvarious reporter constructs as indicated by bars on the left side ofFIG. 5. The relative reporter activities are indicated by black bars onthe right side (FIG. 5). The data show that while w.t. NRF does notalter the expression of the NRE-promoter, the synthetic fusion proteinNRF-VP16 acts as a transcriptional activator of this construct. Therepressor activity of NRF is not detectable in this assay since thereporter does not contain an NF-_(K)B/rel site. The fusion protein doesnot affect promoters which do not contain NRE-recognition sites.

[0103] 6. NRF Inhibits Transcriptional Activity of NF-_(K)B Promoters:

[0104] Transcriptional activity of NRF was tested using constructsencoding either NRF or a chimaeric protein consisting of NRF and theGAL4 DNA-binding domain. Expression of these chimaeric proteins wascarried out in murine cells harbouring the indicated reporterconstructs. These contain GAL4 and NF-_(K)B binding sites as indicated.The relative reporter activities are given as black bars on the rightside (FIG. 6).

[0105] The fusion protein GAL4-NRF exhibits its repressor activitycombined with the DNA-binding property of GAL4. The promoter in which anNF-_(K)B site enhances the basal promoter activity is inhibited by thefusion protein expression, whereas the basal promoter is not affected.W.t. NRF does not affect the reporter gene expression since its promoterdoes not contain an NRE.

[0106] 7. NRF Contains Separable Domains for Silencing and DNA-binding.

[0107] The 100 C-terminal amino acids of NRF are sufficient to bind toNRE. This was demonstrated by expression in E. coli of an incompletecDNA clone. The experiment was as outlined in FIG. 1A.

[0108] In order to define the repressor domain of NRF constructs weredesigned encoding chimaeric proteins containing C-terminal deletions ofNRF and the GAL4 DNA-binding domain. These chimaeric proteins wereexpressed in murine cells harbouring a reporter construct containingGAL4 and NF-_(K)B binding sites as indicated in FIG. 8. The relativereporter activities are indicated by black bars.

[0109] 8. Antisense Expression of NRF Affects Endogenous GeneExpression:

[0110] A conditioned expression plasmid encoding human NRF antisensecDNA (bp 1-344, aa 1-114) was stably transferred into murine C243 cells.IFN activity in the supernatant was measured only upon induction of NRFantisense expression. This result indicates that endogenous NRFexpression which represses a constitutive activity of the IFN-β promoteris eliminated by the antisense RNA.

EXAMPLE 2

[0111] An oligonucleotide having the sequence of FIG. 1B can also beobtained by screening material of a public gene library by means of asynthetic primer, having a subsequence according to FIG. 1B, in a mannernon per se and by isolating the oligonucleotide wanted.

[0112] Example

[0113] IRES activity of human NRF 5′UTR was determined by dicistronicreporter plasmids in vivo. These were constructed by using the Renillaluciferase gene and the firefly luciferase gene under thetranscriptional control of the SV40 promoter. The 5′UTR of NRF orPoliovirus was inserted between two cistrons of this plasmid (FIG. 9A).Resulting constructs were transiently expressed in murine C243 cells.This was done to test NRF 5′UTR for IRES activity and secondly, tocompare the efficiency of human NRF 5′UTR with Poliovirus IRES. Theexpression levels of the reporter genes were compared and indicated asrelative gene expression. As shown in FIG. 9B, the efficiency of NRFIRES is 34,7 fold higher than the efficiency of the Poliovirus IRES. Weperformed Northern blot analysis to provide a control for the equaltranscription of bicistronic mRNAs and to exclude transcript startswithin IRES sequences. As shown in FIG. 9, the size and expression levelof both mRNAs is indistinguishable. Thus, translation initiation takesplace from the human NRF 5′UTR sequence element. Furthermore, thestrength of this IRES element is higher than that of all other knownIRES elements. Since the strength of the Polio virus IRES is about ⅓ ofthe cap dependent translation initiation (Dirks et al., 1993), thestrength of the NRF IRES element is higher than that of cáp-dependenttranslation.

REFERENCES

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[0115] Dirks, W., Wirth, M. and Hauser, H., 1993: Bicistronictranscription units for gene expression in mammalian cells. Gene 128,247-249( )

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1 11 1 3736 DNA Homo sapiens CDS (654)..(1817) 1 cagagtaatg acatggttccttccatcctc caaaggtgac caataatagt ttgtaagtat 60 cattatgaac taatgaattttcaacatatt tgatatattt caatccattg ccatcattgt 120 tcttatcgat atttgagttggctcactttg ccagtaagag tctattcaaa ttggcttctg 180 agtccatttg acacaacacctttgatcttt gacagtttcc ttggttttag gtgctagatg 240 atttctcagg ctcaccttagacatttcctg ccacagactt agaatcagcc atttctctaa 300 ggaccctgat tccatttcatgagaaatgat agagaccaca atcaaaacaa gtcatgaatt 360 tatactgata ttttcaattcaaattaaaga tgaggttttt gctaaatttt tttgagttta 420 tatttgtatg tcttatgctgaaaaatcttg tttcctaatt agtaacataa ttattcattt 480 gatgggtaaa tattttagggccgattcttt ggttttatag ccaagatacc ctgttgataa 540 agtcttgtgg gagcaattataagactggct tattttgaag ctttttaaaa aagacatcct 600 tacctgtttt aactgtagattatattaact taaataggta cagcccacgc ttg atg 656 Met 1 gaa aaa att ctc caaatg gct gaa ggt att gat att ggg gag atg cct 704 Glu Lys Ile Leu Gln MetAla Glu Gly Ile Asp Ile Gly Glu Met Pro 5 10 15 tca tat gat ctg gtg ctgtcc aaa cct tcc aaa ggt caa aaa cgc cac 752 Ser Tyr Asp Leu Val Leu SerLys Pro Ser Lys Gly Gln Lys Arg His 20 25 30 ctc tca aca tgt gat ggt caaaat cct cct aaa aag caa gcc ggt tcc 800 Leu Ser Thr Cys Asp Gly Gln AsnPro Pro Lys Lys Gln Ala Gly Ser 35 40 45 aaa ttc cat gcg aga cct cgt tttgag cct gta cat ttt gta gct agt 848 Lys Phe His Ala Arg Pro Arg Phe GluPro Val His Phe Val Ala Ser 50 55 60 65 agt tca aaa gat gaa gga cag gaagat cct tat ggc cct caa aca aaa 896 Ser Ser Lys Asp Glu Gly Gln Glu AspPro Tyr Gly Pro Gln Thr Lys 70 75 80 gag gta aat gaa caa aca cat ttt gccagc atg cca aga gac atc tac 944 Glu Val Asn Glu Gln Thr His Phe Ala SerMet Pro Arg Asp Ile Tyr 85 90 95 caa gat tat act caa gac tct ttc agt atacaa gat ggg aat tct cag 992 Gln Asp Tyr Thr Gln Asp Ser Phe Ser Ile GlnAsp Gly Asn Ser Gln 100 105 110 tat tgt gat tca tca gga ttc att ctc acaaaa gac cag cct gta aca 1040 Tyr Cys Asp Ser Ser Gly Phe Ile Leu Thr LysAsp Gln Pro Val Thr 115 120 125 gcc aac atg tat ttt gac agt ggg aac cctgcc cca agc acc aca tca 1088 Ala Asn Met Tyr Phe Asp Ser Gly Asn Pro AlaPro Ser Thr Thr Ser 130 135 140 145 cag cag gca aac tct cag tca act cctgag cct tca cca tca cag aca 1136 Gln Gln Ala Asn Ser Gln Ser Thr Pro GluPro Ser Pro Ser Gln Thr 150 155 160 ttt ccc gag tct gtg gta gcc gag aagcag tat ttt att gaa aaa tta 1184 Phe Pro Glu Ser Val Val Ala Glu Lys GlnTyr Phe Ile Glu Lys Leu 165 170 175 acg gcg aca atc tgg aag aac ctt tctaat cca gaa atg act tct gga 1232 Thr Ala Thr Ile Trp Lys Asn Leu Ser AsnPro Glu Met Thr Ser Gly 180 185 190 tct gat aaa att aat tat aca tat atgtta act cgt tgt att cag gcg 1280 Ser Asp Lys Ile Asn Tyr Thr Tyr Met LeuThr Arg Cys Ile Gln Ala 195 200 205 tgt aag aca aat cct gag tat ata tatgct cct tta aag gaa att cct 1328 Cys Lys Thr Asn Pro Glu Tyr Ile Tyr AlaPro Leu Lys Glu Ile Pro 210 215 220 225 cct gcc gac atc ccc aaa aat aaaaaa ctt cta act gat ggc tat gct 1376 Pro Ala Asp Ile Pro Lys Asn Lys LysLeu Leu Thr Asp Gly Tyr Ala 230 235 240 tgt gaa gtt aga tgc caa aat atctac tta act aca ggt tat gct ggc 1424 Cys Glu Val Arg Cys Gln Asn Ile TyrLeu Thr Thr Gly Tyr Ala Gly 245 250 255 agc aag aat ggg tcc agg gat cgagct aca gag cta gct gta aaa ctc 1472 Ser Lys Asn Gly Ser Arg Asp Arg AlaThr Glu Leu Ala Val Lys Leu 260 265 270 ttg cag aaa cgt att gaa gtt agagtt gtc cgg cgg aaa ttc aag cat 1520 Leu Gln Lys Arg Ile Glu Val Arg ValVal Arg Arg Lys Phe Lys His 275 280 285 aca ttt gga gag gac ctc gtg gtgtgt cag att ggc atg tcc tcc tat 1568 Thr Phe Gly Glu Asp Leu Val Val CysGln Ile Gly Met Ser Ser Tyr 290 295 300 305 gaa ttt cct cca gct ctg aagcca cca gaa gac ctg gtg gtg ctg ggt 1616 Glu Phe Pro Pro Ala Leu Lys ProPro Glu Asp Leu Val Val Leu Gly 310 315 320 aaa gat gct tcc ggg cag ccaatt ttt aat gct tct gcc aaa cac tgg 1664 Lys Asp Ala Ser Gly Gln Pro IlePhe Asn Ala Ser Ala Lys His Trp 325 330 335 acc aat ttt gtc att aca gaaaat gca aat gat gca att ggt atc ctt 1712 Thr Asn Phe Val Ile Thr Glu AsnAla Asn Asp Ala Ile Gly Ile Leu 340 345 350 aac aat tct gcc tca ttc aacaag atg tca att gaa tac aaa tat gag 1760 Asn Asn Ser Ala Ser Phe Asn LysMet Ser Ile Glu Tyr Lys Tyr Glu 355 360 365 atg atg cca aat cgc aca tggcgt tcg tcg agt gtt ttt aca aga tca 1808 Met Met Pro Asn Arg Thr Trp ArgSer Ser Ser Val Phe Thr Arg Ser 370 375 380 385 ctg ctt agc tgaaggttatggaaccaaga aaacaagtaa acatgcagct 1857 Leu Leu Ser gccgacgagt ttgaaaattcttcaaaaaca cagcccactt atccatctgt caaaagttca 1917 caatgccata caggctcttcacccagagga tctggaaaga agaaagatat aaaggctctg 1977 tagtttatga gaattcttcaaatcccgtgt gcacgctgaa cgacacagct cagtttaacc 2037 gaatgacagt tgagtatgtctatgaaagga tgacaggcct ccgctggaaa tgcaaagtga 2097 ttctagagag tgaagtaattgcagaagcag ttggggtgaa gaaaactgtc aaatataagc 2157 tgctggggaa gctgtgaaaaccctcaaaaa gacccagcaa ctgtcattaa caacttgaag 2217 aaaggagctg ttgaagatgtgatttcaaga aatgaaattc agggccgctc agcagaggag 2277 gcttacaaac agcaaatcaaagaagataat attggaaatc agctgctgag aaagatgggt 2337 tggactggtg gtggtttaggtaaatctggt gagggcatac gggagcctat ctcagtgaaa 2397 gagcagcata acggaagggcttggtctgga tgtagagagg gtgataaaat gccaagagag 2457 atattgaaca gatcatcagaaactacgaaa gctccgagag ccacacagat ttgactttct 2517 ctagagagct gactaatgatgaacggaagc aaatacatca gattgcccag aagtatggtc 2577 ttaagagtaa gtctcatggggtgggccatg ataggtacct agtggtaggt agaaaaagac 2637 ggaaggaaga cctactagatcagctcaaac aggaaggcca agtgggcatt acgagcttgt 2697 tatgcctcaa gcaaattgagatcttactaa tttattttgt aaatgcctaa tgaggtagat 2757 ttttgaatta aagaaatgctacatgttccg gttgcagagt atattcataa gatgtctcac 2817 cttgttcatt tcacatagtggtttattaga tattggaacc taaagaattc tgtccacttg 2877 tattagctta atccagcagatgatattgtg cagttactgt ttgtgtcttt gatattgctg 2937 tgtccctcag attttagtagtttgacaagc aagaacacat atccaaatgg aattttaccc 2997 tgagaaatta gcattttaaagggcatagca cagcaatctg caacaatatg taaagttgat 3057 attgactaca ataaaaatccagtcttaatt ccagatttac tgaaaatgtc agatcatttt 3117 gtattaatct attttcatctttgtgtgaag ccagttatag aatgtttgac aataaattgt 3177 gctgtacatg tccttaccaacaaatgatgt aaaactttct taaagtaatt ttagtgttat 3237 ttatttataa cttctaccatgtgatttcca gactattgga agtgatttac tgtatcttgt 3297 ggggatatat ttttaacaaattctactctt cacgctgaga gagcactact tgagagagca 3357 gttgaaagtt tcaaaaactttggttcaatc tgaagaaagg aagcttgaac tgtttgttct 3417 tggtgccttg cagagagactcacagcaact ctccattata gctttcacac ggtttggatg 3477 tgcagcacat ccaaggcacaccacagctgt ggtagagctt ggtaaaagac tgaatacatt 3537 ggtgctttga tgaaaaggtcagttggctgg tccctctctc aaaaagctta ttaagcctga 3597 aaagccaact ttgtaacatatttaaaactg ctattttcgc ttatttctgg aatgtaaaaa 3657 aaaaatgtat aaaaagaattagtgtatgct tcctgaataa aaaggagcca aagttgatca 3717 gaaaaaaaaa aaaaaaaaa3736 2 388 PRT Homo sapiens 2 Met Glu Lys Ile Leu Gln Met Ala Glu GlyIle Asp Ile Gly Glu Met 1 5 10 15 Pro Ser Tyr Asp Leu Val Leu Ser LysPro Ser Lys Gly Gln Lys Arg 20 25 30 His Leu Ser Thr Cys Asp Gly Gln AsnPro Pro Lys Lys Gln Ala Gly 35 40 45 Ser Lys Phe His Ala Arg Pro Arg PheGlu Pro Val His Phe Val Ala 50 55 60 Ser Ser Ser Lys Asp Glu Gly Gln GluAsp Pro Tyr Gly Pro Gln Thr 65 70 75 80 Lys Glu Val Asn Glu Gln Thr HisPhe Ala Ser Met Pro Arg Asp Ile 85 90 95 Tyr Gln Asp Tyr Thr Gln Asp SerPhe Ser Ile Gln Asp Gly Asn Ser 100 105 110 Gln Tyr Cys Asp Ser Ser GlyPhe Ile Leu Thr Lys Asp Gln Pro Val 115 120 125 Thr Ala Asn Met Tyr PheAsp Ser Gly Asn Pro Ala Pro Ser Thr Thr 130 135 140 Ser Gln Gln Ala AsnSer Gln Ser Thr Pro Glu Pro Ser Pro Ser Gln 145 150 155 160 Thr Phe ProGlu Ser Val Val Ala Glu Lys Gln Tyr Phe Ile Glu Lys 165 170 175 Leu ThrAla Thr Ile Trp Lys Asn Leu Ser Asn Pro Glu Met Thr Ser 180 185 190 GlySer Asp Lys Ile Asn Tyr Thr Tyr Met Leu Thr Arg Cys Ile Gln 195 200 205Ala Cys Lys Thr Asn Pro Glu Tyr Ile Tyr Ala Pro Leu Lys Glu Ile 210 215220 Pro Pro Ala Asp Ile Pro Lys Asn Lys Lys Leu Leu Thr Asp Gly Tyr 225230 235 240 Ala Cys Glu Val Arg Cys Gln Asn Ile Tyr Leu Thr Thr Gly TyrAla 245 250 255 Gly Ser Lys Asn Gly Ser Arg Asp Arg Ala Thr Glu Leu AlaVal Lys 260 265 270 Leu Leu Gln Lys Arg Ile Glu Val Arg Val Val Arg ArgLys Phe Lys 275 280 285 His Thr Phe Gly Glu Asp Leu Val Val Cys Gln IleGly Met Ser Ser 290 295 300 Tyr Glu Phe Pro Pro Ala Leu Lys Pro Pro GluAsp Leu Val Val Leu 305 310 315 320 Gly Lys Asp Ala Ser Gly Gln Pro IlePhe Asn Ala Ser Ala Lys His 325 330 335 Trp Thr Asn Phe Val Ile Thr GluAsn Ala Asn Asp Ala Ile Gly Ile 340 345 350 Leu Asn Asn Ser Ala Ser PheAsn Lys Met Ser Ile Glu Tyr Lys Tyr 355 360 365 Glu Met Met Pro Asn ArgThr Trp Arg Ser Ser Ser Val Phe Thr Arg 370 375 380 Ser Leu Leu Ser 3853 3736 DNA Mus musculus CDS (654)..(1817) misc_feature (1008) N = A or Cor G or T 3 cagagtaatg acatggttcc ttccatcctc caaaggtgac caataatagtttgtaagtat 60 cattatgaac taatgaattt tcaacatatt tgatatattt caatccattgccatcattgt 120 tcttatcgat atttgagttg gctcactttg ccagtaagag tctattcaaattggcttctg 180 agtccatttg acacaacacc tttgatcttt gacagtttcc ttggttttaggtgctagatg 240 atttctcagg ctcaccttag acatttcctg ccacagactt agaatcagccatttctctaa 300 ggaccctgat tccatttcat gagaaatgat agagaccaca atcaaaacaagtcatgaatt 360 tatactgata ttttcaattc aaattaaaga tgaggttttt gctaaatttttttgagttta 420 tatttgtatg tcttatgctg aaaaatcttg tttcctaatt agtaacataattattcattt 480 gatgggtaaa tattttaggg ccgattcttt ggttttatag ccaagataccctgttgataa 540 agtcttgtgg gagcaattat aagactggct tattttgaag ctttttaaaaaagacatcct 600 tacctgtttt aactgtagat tatattaact taaataggta cagcccacgcttg atg 656 Met 1 gaa aaa att ctc caa atg gct gaa ggt att gat att ggggag atg cct 704 Glu Lys Ile Leu Gln Met Ala Glu Gly Ile Asp Ile Gly GluMet Pro 5 10 15 tca tat gat ctg gtg ctg tcc aaa cct tcc aaa ggt caa aaacgc cac 752 Ser Tyr Asp Leu Val Leu Ser Lys Pro Ser Lys Gly Gln Lys ArgHis 20 25 30 ctc tca aca tgt gat ggt caa aat cct cct aaa aag caa gcc ggttcc 800 Leu Ser Thr Cys Asp Gly Gln Asn Pro Pro Lys Lys Gln Ala Gly Ser35 40 45 aaa ttc cat gcg aga cct cgt ttt gag cct gta cat ttt gta gct agt848 Lys Phe His Ala Arg Pro Arg Phe Glu Pro Val His Phe Val Ala Ser 5055 60 65 agt tca aaa gat gaa gga cag gaa gat cct tat ggc cct caa aca aaa896 Ser Ser Lys Asp Glu Gly Gln Glu Asp Pro Tyr Gly Pro Gln Thr Lys 7075 80 gag gta aat gaa caa aca cat ttt gcc agc atg cca aga gac atc tac944 Glu Val Asn Glu Gln Thr His Phe Ala Ser Met Pro Arg Asp Ile Tyr 8590 95 caa gat tat act caa gac tct ttc agt ata caa gat ggg aat tct caa992 Gln Asp Tyr Thr Gln Asp Ser Phe Ser Ile Gln Asp Gly Asn Ser Gln 100105 110 tat tgt aat tca tca nga ttt att ttc aca aaa gac cag cct gta nca1040 Tyr Cys Asn Ser Ser Xaa Phe Ile Phe Thr Lys Asp Gln Pro Val Xaa 115120 125 acc aac atg tat ttt gac agt ggg aan cct gnc cca agc acc aca tca1088 Thr Asn Met Tyr Phe Asp Ser Gly Xaa Pro Xaa Pro Ser Thr Thr Ser 130135 140 145 cag cag gca aac tct cag tca act cct gag cct tca cca tca cagaca 1136 Gln Gln Ala Asn Ser Gln Ser Thr Pro Glu Pro Ser Pro Ser Gln Thr150 155 160 ttt ccc gag tct gtg gta gcc gag aag cag tat ttt att gaa aaatta 1184 Phe Pro Glu Ser Val Val Ala Glu Lys Gln Tyr Phe Ile Glu Lys Leu165 170 175 acg gcg aca atc tgg aag aac ctt tct aat cca gaa atg act tctgga 1232 Thr Ala Thr Ile Trp Lys Asn Leu Ser Asn Pro Glu Met Thr Ser Gly180 185 190 tct gat aaa att aat tat aca tat atg tta act cgt tgt att caggcg 1280 Ser Asp Lys Ile Asn Tyr Thr Tyr Met Leu Thr Arg Cys Ile Gln Ala195 200 205 tgt aag aca aat cct gag tat ata tat gct cct tta aag gaa attcct 1328 Cys Lys Thr Asn Pro Glu Tyr Ile Tyr Ala Pro Leu Lys Glu Ile Pro210 215 220 225 cct gcc gac atc ccc aaa aat aaa aaa ctt cta act gat ggctat gct 1376 Pro Ala Asp Ile Pro Lys Asn Lys Lys Leu Leu Thr Asp Gly TyrAla 230 235 240 tgt gaa gtt aga tgc caa aat atc tac tta act aca ggt tatgct ggc 1424 Cys Glu Val Arg Cys Gln Asn Ile Tyr Leu Thr Thr Gly Tyr AlaGly 245 250 255 agc aag aat ggg tcc agg gat cga gct aca gag cta gct gtaaaa ctc 1472 Ser Lys Asn Gly Ser Arg Asp Arg Ala Thr Glu Leu Ala Val LysLeu 260 265 270 ttg cag aaa cgt att gaa gtt aga gtt gtc cgg cgg aaa ttcaag cat 1520 Leu Gln Lys Arg Ile Glu Val Arg Val Val Arg Arg Lys Phe LysHis 275 280 285 aca ttt gga gag gac ctc gtg gtg tgt cag att ggc atg tcctcc tat 1568 Thr Phe Gly Glu Asp Leu Val Val Cys Gln Ile Gly Met Ser SerTyr 290 295 300 305 gaa ttt cct cca gct ctg aag cca cca gaa gac ctg gtggtg ctg ggt 1616 Glu Phe Pro Pro Ala Leu Lys Pro Pro Glu Asp Leu Val ValLeu Gly 310 315 320 aaa gat gct tcc ggg cag cca att ttt aat gct tct gccaaa cac tgg 1664 Lys Asp Ala Ser Gly Gln Pro Ile Phe Asn Ala Ser Ala LysHis Trp 325 330 335 acc aat ttt gtc att aca gaa aat gca aat gat gca attggt atc ctt 1712 Thr Asn Phe Val Ile Thr Glu Asn Ala Asn Asp Ala Ile GlyIle Leu 340 345 350 aac aat tct gcc tca ttc aac aag atg tca att gaa tacaaa tat gag 1760 Asn Asn Ser Ala Ser Phe Asn Lys Met Ser Ile Glu Tyr LysTyr Glu 355 360 365 atg atg cca aat cgc aca tgg cgt tcg tcg agt gtt tttaca aga tca 1808 Met Met Pro Asn Arg Thr Trp Arg Ser Ser Ser Val Phe ThrArg Ser 370 375 380 385 ctg ctt agc tgaaggttat ggaaccaaga aaacaagtaaacatgcagct 1857 Leu Leu Ser gccgacgagt ttgaaaattc ttcaaaaaca cagcccacttatccatctgt caaaagttcc 1917 cagtgtcact caggcttttc acccaaagga tccggaaagaagaaagatat caaagatctt 1977 gtgtttatga gaattcttca aatcccgtgt gcacgctgaacgacacagct cagtttaacc 2037 gaatgacagt tgagtatgtc tatgaaagga tgacaggcctccgctggaaa tgcaaagtga 2097 ttctagagag tgaagtaatt gcagaagcag ttggggtgaagaaaactgtc aaatataagc 2157 tgctggggaa gctgtgaaaa ccctcaaaaa gacccagcaactgtcattaa caacttgaag 2217 aaaggagctg ttgaagatgt gatttcaaga aatgaaattcagggccgctc agcagaggag 2277 gcttacaaac agcaaatcaa agaagataat attggaaatcagctgctgag aaagatgggt 2337 tggactggtg gtggtttagg taaatctggt gagggcatacgggagcctat ctcagtgaaa 2397 gagcagcata acggaagggc ttggtctgga tgtagagagggtgataaaat gccaagagag 2457 atattgaaca gatcatcaga aactacgaaa gctccgagagccacacagat ttgactttct 2517 ctagagagct gactaatgat gaacggaagc aaatacatcagattgcccag aagtatggtc 2577 ttaagagtaa gtctcatggg gtgggccatg ataggtacctagtggtaggt agaaaaagac 2637 ggaaggaaga cctactagat cagctcaaac aggaaggccaagtgggcatt acgagcttgt 2697 tatgcctcaa gcaaattgag atcttactaa tttattttgtaaatgcctaa tgaggtagat 2757 ttttgaatta aagaaatgct acatgttccg gttgcagagtatattcataa gatgtctcac 2817 cttgttcatt tcacatagtg gtttattaga tattggaacctaaagaattc tgtccacttg 2877 tattagctta atccagcaga tgatattgtg cagttactgtttgtgtcttt gatattgctg 2937 tgtccctcag attttagtag tttgacaagc aagaacacatatccaaatgg aattttaccc 2997 tgagaaatta gcattttaaa gggcatagca cagcaatctgcaacaatatg taaagttgat 3057 attgactaca ataaaaatcc agtcttaatt ccagatttactgaaaatgtc agatcatttt 3117 gtattaatct attttcatct ttgtgtgaag ccagttatagaatgtttgac aataaattgt 3177 gctgtacatg tccttaccaa caaatgatgt aaaactttcttaaagtaatt ttagtgttat 3237 ttatttataa cttctaccat gtgatttcca gactattggaagtgatttac tgtatcttgt 3297 ggggatatat ttttaacaaa ttctactctt cacgctgagagagcactact tgagagagca 3357 gttgaaagtt tcaaaaactt tggttcaatc tgaagaaaggaagcttgaac tgtttgttct 3417 tggtgccttg cagagagact cacagcaact ctccattatagctttcacac ggtttggatg 3477 tgcagcacat ccaaggcaca ccacagctgt ggtagagcttggtaaaagac tgaatacatt 3537 ggtgctttga tgaaaaggtc agttggctgg tccctctctcaaaaagctta ttaagcctga 3597 aaagccaact ttgtaacata tttaaaactg ctattttcgcttatttctgg aatgtaaaaa 3657 aaaaatgtat aaaaagaatt agtgtatgct tcctgaataaaaaggagcca aagttgatca 3717 gaaaaaaaaa aaaaaaaaa 3736 4 388 PRT Musmusculus misc_feature 119 Xaa = any or unknown amino acid 4 Met Glu LysIle Leu Gln Met Ala Glu Gly Ile Asp Ile Gly Glu Met 1 5 10 15 Pro SerTyr Asp Leu Val Leu Ser Lys Pro Ser Lys Gly Gln Lys Arg 20 25 30 His LeuSer Thr Cys Asp Gly Gln Asn Pro Pro Lys Lys Gln Ala Gly 35 40 45 Ser LysPhe His Ala Arg Pro Arg Phe Glu Pro Val His Phe Val Ala 50 55 60 Ser SerSer Lys Asp Glu Gly Gln Glu Asp Pro Tyr Gly Pro Gln Thr 65 70 75 80 LysGlu Val Asn Glu Gln Thr His Phe Ala Ser Met Pro Arg Asp Ile 85 90 95 TyrGln Asp Tyr Thr Gln Asp Ser Phe Ser Ile Gln Asp Gly Asn Ser 100 105 110Gln Tyr Cys Asn Ser Ser Xaa Phe Ile Phe Thr Lys Asp Gln Pro Val 115 120125 Xaa Thr Asn Met Tyr Phe Asp Ser Gly Xaa Pro Xaa Pro Ser Thr Thr 130135 140 Ser Gln Gln Ala Asn Ser Gln Ser Thr Pro Glu Pro Ser Pro Ser Gln145 150 155 160 Thr Phe Pro Glu Ser Val Val Ala Glu Lys Gln Tyr Phe IleGlu Lys 165 170 175 Leu Thr Ala Thr Ile Trp Lys Asn Leu Ser Asn Pro GluMet Thr Ser 180 185 190 Gly Ser Asp Lys Ile Asn Tyr Thr Tyr Met Leu ThrArg Cys Ile Gln 195 200 205 Ala Cys Lys Thr Asn Pro Glu Tyr Ile Tyr AlaPro Leu Lys Glu Ile 210 215 220 Pro Pro Ala Asp Ile Pro Lys Asn Lys LysLeu Leu Thr Asp Gly Tyr 225 230 235 240 Ala Cys Glu Val Arg Cys Gln AsnIle Tyr Leu Thr Thr Gly Tyr Ala 245 250 255 Gly Ser Lys Asn Gly Ser ArgAsp Arg Ala Thr Glu Leu Ala Val Lys 260 265 270 Leu Leu Gln Lys Arg IleGlu Val Arg Val Val Arg Arg Lys Phe Lys 275 280 285 His Thr Phe Gly GluAsp Leu Val Val Cys Gln Ile Gly Met Ser Ser 290 295 300 Tyr Glu Phe ProPro Ala Leu Lys Pro Pro Glu Asp Leu Val Val Leu 305 310 315 320 Gly LysAsp Ala Ser Gly Gln Pro Ile Phe Asn Ala Ser Ala Lys His 325 330 335 TrpThr Asn Phe Val Ile Thr Glu Asn Ala Asn Asp Ala Ile Gly Ile 340 345 350Leu Asn Asn Ser Ala Ser Phe Asn Lys Met Ser Ile Glu Tyr Lys Tyr 355 360365 Glu Met Met Pro Asn Arg Thr Trp Arg Ser Ser Ser Val Phe Thr Arg 370375 380 Ser Leu Leu Ser 385 5 1164 DNA Homo sapiens CDS (1)..(1164) 5atg gaa aaa att ctc caa atg gct gaa ggt att gat att ggg gag atg 48 MetGlu Lys Ile Leu Gln Met Ala Glu Gly Ile Asp Ile Gly Glu Met 1 5 10 15cct tca tat gat ctg gtg ctg tcc aaa cct tcc aaa ggt caa aaa cgc 96 ProSer Tyr Asp Leu Val Leu Ser Lys Pro Ser Lys Gly Gln Lys Arg 20 25 30 cacctc tca aca tgt gat ggt caa aat cct cct aaa aag caa gcc ggt 144 His LeuSer Thr Cys Asp Gly Gln Asn Pro Pro Lys Lys Gln Ala Gly 35 40 45 tcc aaattc cat gcg aga cct cgt ttt gag cct gta cat ttt gta gct 192 Ser Lys PheHis Ala Arg Pro Arg Phe Glu Pro Val His Phe Val Ala 50 55 60 agt agt tcaaaa gat gaa gga cag gaa gat cct tat ggc cct caa aca 240 Ser Ser Ser LysAsp Glu Gly Gln Glu Asp Pro Tyr Gly Pro Gln Thr 65 70 75 80 aaa gag gtaaat gaa caa aca cat ttt gcc agc atg cca aga gac atc 288 Lys Glu Val AsnGlu Gln Thr His Phe Ala Ser Met Pro Arg Asp Ile 85 90 95 tac caa gat tatact caa gac tct ttc agt ata caa gat ggg aat tct 336 Tyr Gln Asp Tyr ThrGln Asp Ser Phe Ser Ile Gln Asp Gly Asn Ser 100 105 110 cag tat tgt gattca tca gga ttc att ctc aca aaa gac cag cct gta 384 Gln Tyr Cys Asp SerSer Gly Phe Ile Leu Thr Lys Asp Gln Pro Val 115 120 125 aca gcc aac atgtat ttt gac agt ggg aac cct gcc cca agc acc aca 432 Thr Ala Asn Met TyrPhe Asp Ser Gly Asn Pro Ala Pro Ser Thr Thr 130 135 140 tca cag cag gcaaac tct cag tca act cct gag cct tca cca tca cag 480 Ser Gln Gln Ala AsnSer Gln Ser Thr Pro Glu Pro Ser Pro Ser Gln 145 150 155 160 aca ttt cccgag tct gtg gta gcc gag aag cag tat ttt att gaa aaa 528 Thr Phe Pro GluSer Val Val Ala Glu Lys Gln Tyr Phe Ile Glu Lys 165 170 175 tta acg gcgaca atc tgg aag aac ctt tct aat cca gaa atg act tct 576 Leu Thr Ala ThrIle Trp Lys Asn Leu Ser Asn Pro Glu Met Thr Ser 180 185 190 gga tct gataaa att aat tat aca tat atg tta act cgt tgt att cag 624 Gly Ser Asp LysIle Asn Tyr Thr Tyr Met Leu Thr Arg Cys Ile Gln 195 200 205 gcg tgt aagaca aat cct gag tat ata tat gct cct tta aag gaa att 672 Ala Cys Lys ThrAsn Pro Glu Tyr Ile Tyr Ala Pro Leu Lys Glu Ile 210 215 220 cct cct gccgac atc ccc aaa aat aaa aaa ctt cta act gat ggc tat 720 Pro Pro Ala AspIle Pro Lys Asn Lys Lys Leu Leu Thr Asp Gly Tyr 225 230 235 240 gct tgtgaa gtt aga tgc caa aat atc tac tta act aca ggt tat gct 768 Ala Cys GluVal Arg Cys Gln Asn Ile Tyr Leu Thr Thr Gly Tyr Ala 245 250 255 ggc agcaag aat ggg tcc agg gat cga gct aca gag cta gct gta aaa 816 Gly Ser LysAsn Gly Ser Arg Asp Arg Ala Thr Glu Leu Ala Val Lys 260 265 270 ctc ttgcag aaa cgt att gaa gtt aga gtt gtc cgg cgg aaa ttc aag 864 Leu Leu GlnLys Arg Ile Glu Val Arg Val Val Arg Arg Lys Phe Lys 275 280 285 cat acattt gga gag gac ctc gtg gtg tgt cag att ggc atg tcc tcc 912 His Thr PheGly Glu Asp Leu Val Val Cys Gln Ile Gly Met Ser Ser 290 295 300 tat gaattt cct cca gct ctg aag cca cca gaa gac ctg gtg gtg ctg 960 Tyr Glu PhePro Pro Ala Leu Lys Pro Pro Glu Asp Leu Val Val Leu 305 310 315 320 ggtaaa gat gct tcc ggg cag cca att ttt aat gct tct gcc aaa cac 1008 Gly LysAsp Ala Ser Gly Gln Pro Ile Phe Asn Ala Ser Ala Lys His 325 330 335 tggacc aat ttt gtc att aca gaa aat gca aat gat gca att ggt atc 1056 Trp ThrAsn Phe Val Ile Thr Glu Asn Ala Asn Asp Ala Ile Gly Ile 340 345 350 cttaac aat tct gcc tca ttc aac aag atg tca att gaa tac aaa tat 1104 Leu AsnAsn Ser Ala Ser Phe Asn Lys Met Ser Ile Glu Tyr Lys Tyr 355 360 365 gagatg atg cca aat cgc aca tgg cgt tcg tcg agt gtt ttt aca aga 1152 Glu MetMet Pro Asn Arg Thr Trp Arg Ser Ser Ser Val Phe Thr Arg 370 375 380 tcactg ctt agc 1164 Ser Leu Leu Ser 385 6 388 PRT Homo sapiens 6 Met GluLys Ile Leu Gln Met Ala Glu Gly Ile Asp Ile Gly Glu Met 1 5 10 15 ProSer Tyr Asp Leu Val Leu Ser Lys Pro Ser Lys Gly Gln Lys Arg 20 25 30 HisLeu Ser Thr Cys Asp Gly Gln Asn Pro Pro Lys Lys Gln Ala Gly 35 40 45 SerLys Phe His Ala Arg Pro Arg Phe Glu Pro Val His Phe Val Ala 50 55 60 SerSer Ser Lys Asp Glu Gly Gln Glu Asp Pro Tyr Gly Pro Gln Thr 65 70 75 80Lys Glu Val Asn Glu Gln Thr His Phe Ala Ser Met Pro Arg Asp Ile 85 90 95Tyr Gln Asp Tyr Thr Gln Asp Ser Phe Ser Ile Gln Asp Gly Asn Ser 100 105110 Gln Tyr Cys Asp Ser Ser Gly Phe Ile Leu Thr Lys Asp Gln Pro Val 115120 125 Thr Ala Asn Met Tyr Phe Asp Ser Gly Asn Pro Ala Pro Ser Thr Thr130 135 140 Ser Gln Gln Ala Asn Ser Gln Ser Thr Pro Glu Pro Ser Pro SerGln 145 150 155 160 Thr Phe Pro Glu Ser Val Val Ala Glu Lys Gln Tyr PheIle Glu Lys 165 170 175 Leu Thr Ala Thr Ile Trp Lys Asn Leu Ser Asn ProGlu Met Thr Ser 180 185 190 Gly Ser Asp Lys Ile Asn Tyr Thr Tyr Met LeuThr Arg Cys Ile Gln 195 200 205 Ala Cys Lys Thr Asn Pro Glu Tyr Ile TyrAla Pro Leu Lys Glu Ile 210 215 220 Pro Pro Ala Asp Ile Pro Lys Asn LysLys Leu Leu Thr Asp Gly Tyr 225 230 235 240 Ala Cys Glu Val Arg Cys GlnAsn Ile Tyr Leu Thr Thr Gly Tyr Ala 245 250 255 Gly Ser Lys Asn Gly SerArg Asp Arg Ala Thr Glu Leu Ala Val Lys 260 265 270 Leu Leu Gln Lys ArgIle Glu Val Arg Val Val Arg Arg Lys Phe Lys 275 280 285 His Thr Phe GlyGlu Asp Leu Val Val Cys Gln Ile Gly Met Ser Ser 290 295 300 Tyr Glu PhePro Pro Ala Leu Lys Pro Pro Glu Asp Leu Val Val Leu 305 310 315 320 GlyLys Asp Ala Ser Gly Gln Pro Ile Phe Asn Ala Ser Ala Lys His 325 330 335Trp Thr Asn Phe Val Ile Thr Glu Asn Ala Asn Asp Ala Ile Gly Ile 340 345350 Leu Asn Asn Ser Ala Ser Phe Asn Lys Met Ser Ile Glu Tyr Lys Tyr 355360 365 Glu Met Met Pro Asn Arg Thr Trp Arg Ser Ser Ser Val Phe Thr Arg370 375 380 Ser Leu Leu Ser 385 7 300 DNA Homo sapiens CDS (1)..(300) 7cat aca ttt gga gag gac ctc gtg gtg tgt cag att ggc atg tcc tcc 48 HisThr Phe Gly Glu Asp Leu Val Val Cys Gln Ile Gly Met Ser Ser 1 5 10 15tat gaa ttt cct cca gct ctg aag cca cca gaa gac ctg gtg gtg ctg 96 TyrGlu Phe Pro Pro Ala Leu Lys Pro Pro Glu Asp Leu Val Val Leu 20 25 30 ggtaaa gat gct tcc ggg cag cca att ttt aat gct tct gcc aaa cac 144 Gly LysAsp Ala Ser Gly Gln Pro Ile Phe Asn Ala Ser Ala Lys His 35 40 45 tgg accaat ttt gtc att aca gaa aat gca aat gat gca att ggt atc 192 Trp Thr AsnPhe Val Ile Thr Glu Asn Ala Asn Asp Ala Ile Gly Ile 50 55 60 ctt aac aattct gcc tca ttc aac aag atg tca att gaa tac aaa tat 240 Leu Asn Asn SerAla Ser Phe Asn Lys Met Ser Ile Glu Tyr Lys Tyr 65 70 75 80 gag atg atgcca aat cgc aca tgg cgt tcg tcg agt gtt ttt aca aga 288 Glu Met Met ProAsn Arg Thr Trp Arg Ser Ser Ser Val Phe Thr Arg 85 90 95 tca ctg ctt agc300 Ser Leu Leu Ser 100 8 100 PRT Homo sapiens 8 His Thr Phe Gly Glu AspLeu Val Val Cys Gln Ile Gly Met Ser Ser 1 5 10 15 Tyr Glu Phe Pro ProAla Leu Lys Pro Pro Glu Asp Leu Val Val Leu 20 25 30 Gly Lys Asp Ala SerGly Gln Pro Ile Phe Asn Ala Ser Ala Lys His 35 40 45 Trp Thr Asn Phe ValIle Thr Glu Asn Ala Asn Asp Ala Ile Gly Ile 50 55 60 Leu Asn Asn Ser AlaSer Phe Asn Lys Met Ser Ile Glu Tyr Lys Tyr 65 70 75 80 Glu Met Met ProAsn Arg Thr Trp Arg Ser Ser Ser Val Phe Thr Arg 85 90 95 Ser Leu Leu Ser100 9 873 DNA Homo sapiens CDS (1)..(873) 9 atg gaa aaa att ctc caa atggct gaa ggt att gat att ggg gag atg 48 Met Glu Lys Ile Leu Gln Met AlaGlu Gly Ile Asp Ile Gly Glu Met 1 5 10 15 cct tca tat gat ctg gtg ctgtcc aaa cct tcc aaa ggt caa aaa cgc 96 Pro Ser Tyr Asp Leu Val Leu SerLys Pro Ser Lys Gly Gln Lys Arg 20 25 30 cac ctc tca aca tgt gat ggt caaaat cct cct aaa aag caa gcc ggt 144 His Leu Ser Thr Cys Asp Gly Gln AsnPro Pro Lys Lys Gln Ala Gly 35 40 45 tcc aaa ttc cat gcg aga cct cgt tttgag cct gta cat ttt gta gct 192 Ser Lys Phe His Ala Arg Pro Arg Phe GluPro Val His Phe Val Ala 50 55 60 agt agt tca aaa gat gaa gga cag gaa gatcct tat ggc cct caa aca 240 Ser Ser Ser Lys Asp Glu Gly Gln Glu Asp ProTyr Gly Pro Gln Thr 65 70 75 80 aaa gag gta aat gaa caa aca cat ttt gccagc atg cca aga gac atc 288 Lys Glu Val Asn Glu Gln Thr His Phe Ala SerMet Pro Arg Asp Ile 85 90 95 tac caa gat tat act caa gac tct ttc agt atacaa gat ggg aat tct 336 Tyr Gln Asp Tyr Thr Gln Asp Ser Phe Ser Ile GlnAsp Gly Asn Ser 100 105 110 cag tat tgt gat tca tca gga ttc att ctc acaaaa gac cag cct gta 384 Gln Tyr Cys Asp Ser Ser Gly Phe Ile Leu Thr LysAsp Gln Pro Val 115 120 125 aca gcc aac atg tat ttt gac agt ggg aac cctgcc cca agc acc aca 432 Thr Ala Asn Met Tyr Phe Asp Ser Gly Asn Pro AlaPro Ser Thr Thr 130 135 140 tca cag cag gca aac tct cag tca act cct gagcct tca cca tca cag 480 Ser Gln Gln Ala Asn Ser Gln Ser Thr Pro Glu ProSer Pro Ser Gln 145 150 155 160 aca ttt ccc gag tct gtg gta gcc gag aagcag tat ttt att gaa aaa 528 Thr Phe Pro Glu Ser Val Val Ala Glu Lys GlnTyr Phe Ile Glu Lys 165 170 175 tta acg gcg aca atc tgg aag aac ctt tctaat cca gaa atg act tct 576 Leu Thr Ala Thr Ile Trp Lys Asn Leu Ser AsnPro Glu Met Thr Ser 180 185 190 gga tct gat aaa att aat tat aca tat atgtta act cgt tgt att cag 624 Gly Ser Asp Lys Ile Asn Tyr Thr Tyr Met LeuThr Arg Cys Ile Gln 195 200 205 gcg tgt aag aca aat cct gag tat ata tatgct cct tta aag gaa att 672 Ala Cys Lys Thr Asn Pro Glu Tyr Ile Tyr AlaPro Leu Lys Glu Ile 210 215 220 cct cct gcc gac atc ccc aaa aat aaa aaactt cta act gat ggc tat 720 Pro Pro Ala Asp Ile Pro Lys Asn Lys Lys LeuLeu Thr Asp Gly Tyr 225 230 235 240 gct tgt gaa gtt aga tgc caa aat atctac tta act aca ggt tat gct 768 Ala Cys Glu Val Arg Cys Gln Asn Ile TyrLeu Thr Thr Gly Tyr Ala 245 250 255 ggc agc aag aat ggg tcc agg gat cgagct aca gag cta gct gta aaa 816 Gly Ser Lys Asn Gly Ser Arg Asp Arg AlaThr Glu Leu Ala Val Lys 260 265 270 ctc ttg cag aaa cgt att gaa gtt agagtt gtc cgg cgg aaa ttc aag 864 Leu Leu Gln Lys Arg Ile Glu Val Arg ValVal Arg Arg Lys Phe Lys 275 280 285 cat aca ttt 873 His Thr Phe 290 10291 PRT Homo sapiens 10 Met Glu Lys Ile Leu Gln Met Ala Glu Gly Ile AspIle Gly Glu Met 1 5 10 15 Pro Ser Tyr Asp Leu Val Leu Ser Lys Pro SerLys Gly Gln Lys Arg 20 25 30 His Leu Ser Thr Cys Asp Gly Gln Asn Pro ProLys Lys Gln Ala Gly 35 40 45 Ser Lys Phe His Ala Arg Pro Arg Phe Glu ProVal His Phe Val Ala 50 55 60 Ser Ser Ser Lys Asp Glu Gly Gln Glu Asp ProTyr Gly Pro Gln Thr 65 70 75 80 Lys Glu Val Asn Glu Gln Thr His Phe AlaSer Met Pro Arg Asp Ile 85 90 95 Tyr Gln Asp Tyr Thr Gln Asp Ser Phe SerIle Gln Asp Gly Asn Ser 100 105 110 Gln Tyr Cys Asp Ser Ser Gly Phe IleLeu Thr Lys Asp Gln Pro Val 115 120 125 Thr Ala Asn Met Tyr Phe Asp SerGly Asn Pro Ala Pro Ser Thr Thr 130 135 140 Ser Gln Gln Ala Asn Ser GlnSer Thr Pro Glu Pro Ser Pro Ser Gln 145 150 155 160 Thr Phe Pro Glu SerVal Val Ala Glu Lys Gln Tyr Phe Ile Glu Lys 165 170 175 Leu Thr Ala ThrIle Trp Lys Asn Leu Ser Asn Pro Glu Met Thr Ser 180 185 190 Gly Ser AspLys Ile Asn Tyr Thr Tyr Met Leu Thr Arg Cys Ile Gln 195 200 205 Ala CysLys Thr Asn Pro Glu Tyr Ile Tyr Ala Pro Leu Lys Glu Ile 210 215 220 ProPro Ala Asp Ile Pro Lys Asn Lys Lys Leu Leu Thr Asp Gly Tyr 225 230 235240 Ala Cys Glu Val Arg Cys Gln Asn Ile Tyr Leu Thr Thr Gly Tyr Ala 245250 255 Gly Ser Lys Asn Gly Ser Arg Asp Arg Ala Thr Glu Leu Ala Val Lys260 265 270 Leu Leu Gln Lys Arg Ile Glu Val Arg Val Val Arg Arg Lys PheLys 275 280 285 His Thr Phe 290 11 653 DNA Homo sapiens 11 cagagtaatgacatggttcc ttccatcctc caaaggtgac caataatagt ttgtaagtat 60 cattatgaactaatgaattt tcaacatatt tgatatattt caatccattg ccatcattgt 120 tcttatcgatatttgagttg gctcactttg ccagtaagag tctattcaaa ttggcttctg 180 agtccatttgacacaacacc tttgatcttt gacagtttcc ttggttttag gtgctagatg 240 atttctcaggctcaccttag acatttcctg ccacagactt agaatcagcc atttctctaa 300 ggaccctgattccatttcat gagaaatgat agagaccaca atcaaaacaa gtcatgaatt 360 tatactgatattttcaattc aaattaaaga tgaggttttt gctaaatttt tttgagttta 420 tatttgtatgtcttatgctg aaaaatcttg tttcctaatt agtaacataa ttattcattt 480 gatgggtaaatattttaggg ccgattcttt ggttttatag ccaagatacc ctgttgataa 540 agtcttgtgggagcaattat aagactggct tattttgaag ctttttaaaa aagacatcct 600 tacctgttttaactgtagat tatattaact taaataggta cagcccacgc ttg 653

What is claimed is:
 1. An isolated single stranded polynucleotidecomprising a polynucleotide sequence selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, fragments thereof, and polynucleotides complementarythereto.
 2. An isolated single stranded polynucleotide comprising apolynucleotide sequence set forth in SEQ ID NO: 1 wherein the singlestranded polynucleotide is hybridizable to a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11, at a temperature of atleast 25° C. and a 1M sodium chloride concentration.
 3. An isolatedsingle stranded polynucleotide comprising a polynucleotide sequence setforth in SEQ ID NO: 3 wherein the single stranded polynucleotide ishybridizable to a polynucleotide comprising a polynucleotide sequenceselected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ IDNO: 9, and SEQ ID NO: 11, at a temperature of at least 25° C. and a 1Msodium chloride concentration.
 4. A double stranded polynucleotidecomprising a single stranded polynucleotide according to claim 1 and itscomplementary strand.
 5. A double stranded polynucleotide comprising asingle stranded polynucleotide according to claim 2 and itscomplementary strand.
 6. A double stranded polynucleotide comprising asingle stranded polynucleotide according to claim 3 and itscomplementary strand.
 7. An RNA (a) comprising a sequence complementaryto a polynucleotide according to claim 1, (b) according to (a) where theRNA is anti-sense RNA, or (c) being a degradation product of a RNAaccording to (a) or (b).
 8. An RNA (a) comprising a sequencecomplementary to a polynucleotide according to claim 2, (b) according to(a) where the RNA is anti-sense RNA, or (c) being a degradation productof a RNA according to (a) or (b).
 9. An RNA (a) comprising a sequencecomplementary to a polynucleotide according to claim 3, (b) according to(a) where the RNA is anti-sense RNA, or (c) being a degradation productof a RNA according to (a) or (b).
 10. An RNA (a) comprising a sequencecomplementary to a polynucleotide according to claim 4, (b) according to(a) where the RNA is anti-sense RNA, or (c) being a degradation productof a RNA according to (a) or (b).
 11. An RNA (a) comprising a sequencecomplementary to a polynucleotide according to claim 5, (b) according to(a) where the RNA is anti-sense RNA, or (c) being a degradation productof a RNA according to (a) or (b).
 12. An RNA (a) comprising a sequencecomplementary to a polynucleotide according to claim 6, (b) according to(a) where the RNA is anti-sense RNA, or (c) being a degradation productof a RNA according to (a) or (b).
 13. A vector which comprises a doublestranded polynucleotide according to claim
 4. 14. The vector accordingto claim 13 wherein the double stranded polynucleotide encodes apolypeptide in antisense direction.
 15. A vector which comprises adouble stranded polynucleotide according to claim
 5. 16. The vectoraccording to claim 15 wherein the double stranded polynucleotide encodesa polypeptide in antisense direction.
 17. A vector which comprises adouble stranded polynucleotide according to claim
 6. 18. The vectoraccording to claim 17 wherein the double stranded polynucleotide encodesa polypeptide in antisense direction.
 19. A recombinant host cellcomprising a polynucleotide selected from the group consisting of: (a) apolynucleotide according to claim 1; (b) a polynucleotide comprising apolynucleotide sequence encoding the polypeptide as set forth in SEQ IDNO: 2; (c) a polynucleotide comprising a polynucleotide sequence whichhybridizes to the complement of either of (a) or (b); and (d) a doublestranded polynucleotide comprising a single stranded polynucleotideaccording to (a), (b) or (c) and its complementary strand.
 20. A methodfor producing an NRF polypeptide comprising the steps of: (i) culturinga host cell according to claim 19 in growth medium under conditionssuitable for expression of the NRF polypeptide; and (ii) isolating theexpressed NRF polypeptide from the cell or the medium.
 21. The NRFpolypeptide of claim 20, fragments thereof, and variants thereof.
 22. AnNRF polypeptide selected from the group consisting of SEQ ID NO:
 2. SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, fragments thereof,and variants thereof.
 23. The polypeptide according to claim 22 furthercomprising another functional polypeptide or functional fragment thereoffused thereto.
 24. The polypeptide according to claim 22 wherein thepolypeptide is an unfused polypeptide.
 25. A method of screening for adominant negative mutant NRF polypeptide comprising: (i) mutating thepolynucleotide sequence of a single stranded polynucleotide according toclaim 1 to a make a mutated single stranded polynucleotide; (ii)expressing the mutated single stranded polynucleotide to produce anexpression product; (iii) subjecting the expression product to acompeting test for inhibition of transcription with a polypeptideencoded by an unmodified single stranded polynucleotide; and (iv)identifying as a dominant negative mutant NRF polypeptide an expressionproduct that competed with the polypeptide encoded by the unmodifiedsingle stranded polynucleotide in the competing test of (c).
 26. Adominant negative mutant protein identified in claim
 25. 27. A method ofidentifying an antagonist of NRF polypeptide binding to a polynucleotidecomprising the steps of: (i) exposing an NRF polypeptide according toclaim 22 to a polynucleotide under conditions which permit binding ofNRF polypeptide to a polynucleotide in the presence and absence of atest compound; (ii) measuring the binding of NRF polypeptide to apolynucleotide in the presence and absence of the test compound; and(iii) identifying as antagonist a test compound by its ability toprevent binding of NRF polypeptide to a polynucleotide.
 28. A method ofidentifying an agonist of NRF polypeptide binding to a polynucleotidecomprising the steps of: (i) exposing an NRF polypeptide according toclaim 22 to a polynucleotide encoding a polypeptide under conditionswhich permit binding of NRF polypeptide to a polynucleotide in thepresence and absence of a test compound; (ii) measuring the polypeptideproduced by binding of the test compound to the polynucleotide; and(iii) identifying as agonist a test compound by its ability to furtherreduce polypeptide production in the presence as opposed to the absenceof the test compound.
 29. A method of preventing NRF polypeptideexpression comprising introducing a polynucleotide according to claim 1to a eucaryotic cell or a transgenic animal, wherein the polynucleotideencodes an NRF polypeptide in antisense.
 30. A method of preventing NRFpolypeptide expression comprising introducing an RNA (a) comprising asequence complementary to a polynucleotide according to claim 1, (b)according to (a) where the RNA is antisense RNA, or (c) being adegradation product of an RNA according to (a) or (b) to a eucaryoticcell or a transgenic animal.
 31. A ribozyme comprising an RNA (a)comprising a sequence complementary to a polynucleotide according toclaim 1, (b) according to (a) where the RNA is antisense RNA, or (c)being a degradation product of an RNA according to (a) or (b).
 32. Amethod of detecting and diagnosing transient or permanent regulatorydisorders of NF_(K)B/rel- and/or NRF-regulated physiological patterns inanimals comprising: (i) determining the presence or amount of expressionof an NRF polypeptide according to claim 22 in a biological sample; and(ii) diagnosing transient or permanent regulatory disorders ofNF_(K)B/rel- and/or NRF-regulated physiological patterns based on thepresence or amount of expression of the NRF polypeptide.
 33. A method oftreating or ameliorating a disease selected from the group consisting ofrheumatoid arthritis, inflammations, infectious diseases, tumors, andgenetic diseases, the method comprising administering a polynucleotideof claim 1 in an effective amount.
 34. A method of treating orameliorating a disease selected from the group consisting of rheumatoidarthritis, inflammations, infectious diseases, tumors, and geneticdiseases. the method comprising administering a polynucleotide of claim4 in an effective amount.
 35. A method of treating or ameliorating adisease selected from the group consisting of rheumatoid arthritis,inflammations, infectious diseases, tumors, and genetic diseases. themethod comprising administering a polypeptide of claim 22 in aneffective amount.
 36. A method of gene therapy in animals comprisingadministering to an animal a polynucleotide of claim
 1. 37. A method ofgene therapy in animals comprising administering to an animal apolynucleotide of claim
 4. 38. An RNA comprising a sequencecomplementary to a polynucleotide (a) comprising a polynucleotidesequence set forth in SEQ ID No. 11, (b) according to (a) where the RNAis anti-sense RNA, or (c) being a degradation product of an RNAaccording to (a) or (b).
 39. A method of expressing an open readingframe in a eucaryotic cell or a transgenic animal comprising: (i)inserting an RNA according to claim 38 as an internal ribosome entrysite element in a polycistronic expression vector containing two or moreopen reading frames; and (ii) introducing the polycistronic expressionvector to the eucaryotic cell or the transgenic animal.
 40. Apolycistronic expression vector which comprises two or more open readingframes and an RNA according to claim 38, wherein the RNA serves as aninternal ribosome entry site.
 41. A method of enhancing translation inan eucaryotic cell or a transgenic animal comprising: (i) inserting anRNA according to claim 38 as a translational enhancer in a monocistronicexpression vector or a polycistronic expression vector; and (ii)introducing the monocistronic expression vector or polycistronicexpression vector to the eucaryotic cell or the transgenic animal.
 42. Amonocistronic expression vector or a polycistronic expression vectorwhich comprises one or more open reading frames and an RNA according toclaim 38, wherein the RNA serves as a translational enhancer.